BackgroundLocal inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.Methodology/Principal FindingsWe demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8+ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.Conclusions/SignificanceOur findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.

The cancer stem cell (CSC) hypothesis has gained significant recognition as a descriptor of tumorigenesis. Additionally, tumor-associated macrophages (TAMs) are known to promote growth and metastasis of breast cancer. However, it is not known whether TAMs mediate tumorigenesis through regulation of breast CSCs. Here, we report that TAMs promote CSC-like phenotypes in murine breast cancer cells by upregulating their expression of Sox-2. These CSC-like phenotypes were characterized by increased Sox-2, Oct-4, Nanog, AbcG2, and Sca-1 gene expression, in addition to increased drug-efflux capacity, resistance to chemotherapy, and increased tumorigenicity in vivo. Downregulation of Sox-2 in tumor cells by siRNA blocked the ability of TAMs to induce these CSC-like phenotypes and inhibited tumor growth in vivo. Furthermore, we identified a novel epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (Stat3)/Sox-2 paracrine signaling pathway between macrophages and mouse breast cancer cells that is required for macrophage-induced upregulation of Sox-2 and CSC phenotypes in tumor cells. We showed that this crosstalk was effectively blocked by the small molecule inhibitors AG1478 or CDDO-Im against EGFR and Stat3, respectively. Therefore, our report identifies a novel role for TAMs in breast CSC regulation and establishes a rationale for targeting the EGFR/Stat3/Sox-2 signaling pathway for CSC therapy. STEM CELLS 2013;31:248-258 Disclosure of potential conflicts of interest is found at the end of this article.

Background:The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ‘side population' (SP).Methods and results:The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice.Conclusions:These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

IntroductionAccording to the cancer stem cell hypothesis, tumor initiation and tumor growth are driven by a small population of stem cell-like cancer cells (SCLCCs), which have an indefinite proliferative and differentiation potential. 1,2 The limited number of SCLCCs in the tumor and their specific phenotypes have been made responsible for their escape from conventional therapies resulting in disease relapse. 1,3,4 These SCLCCs have been identified in hematologic tumors, such as leukemia, 5,6 as well as in various solid malignancies such as brain, breast, prostate, and lung cancers. [7][8][9][10][11] SCLCCs have been identified in such tumors, as well as tumor cell lines, based on the expression, or lack thereof, of surface markers such as CD44, CD24, CD29, Lin, CD133, and Sca-1 or by their ability to form spheres in cell culture. 7,8,[11][12][13] Another useful approach for identification and purification of SCLCCs, specifically in the absence of surface marker expression, has been to use the phenomenon that stem cells in general have the ability to efflux lipophilic, fluorescent dyes such as Hoechst 33342. The efflux of the Hoechst 33342 dye has been correlated with ABC transporters, in particular ABCG2, 14 and can be inhibited with Ca ϩϩ -channel blockers. This Hoechst low population has been designated the side population (SP), and it was first shown in the hematopoietic system that SP cells within the bone marrow are greatly enriched for stem cells. 15,16 In addition to the identification of hematopoietic stem cells (HSCs) in the bone marrow, this method has also been used to identify SP cells with stem cell properties in various tissues such as mammary gland, skin, brain, and liver. [17][18][19][20][21] Most recently, this approach was also applied to identify an SCLCC-enriched SP compartment in various human and rat carcinomas. [22][23][24][25] To date, the majority of studies examining SCLCCs isolated these cells from human or rat tumors or tumor cell lines and tested their tumorigenic potential by transplanting them into immunecompromised severe combined immunodeficient (SCID), SCID/ NOD (nonobese diabetic), or nude mice. 7,9,23,24,26 As of late, the clinical relevance of such models has come into question. For example, it has been suggested that xenografts in immunecompromised mice may not recapitulate the full extent of tumor development seen in human cancers. 27 Importantly, the host immune status and the microenvironment both play a crucial role during tumor initiation and development because they can alter expression levels of a wide range of genes in tumor cells. Therefore, xenografts in immune-compromised mice lack a crucial syngeneic interface between tumor and microenvironment, which may reflect on tumor growth and dissemination dynamics. [28][29][30][31] Here, we demonstrate for the first time that the SCLCC concept can also be applied to syngeneic mouse tumor models. To this end, we identified a low Hoechst 33342 dye-retaining SP in murine 4T1 breast carcinoma and NXS2 neuroblastoma cell lin...

A growing body of evidence indicates that interactions between neoplastic cells and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) are crucial in promoting tumor cell invasion and progression. Macrophages play an ambiguous role in these processes since this M1 phenotype correlates with tumoricidal capacity whereas TAMs of M2 phenotype exert tumor-promoting effects. Here, we provide evidence that interactions between mouse breast tumor cells and TAMs remodel the TME, leading to upregulation of Fra-1, a member of the FOS family of transcription factor. In turn, this proto-oncogene initiates activation of the IL-6/JAK/Stat3 signaling pathway. This creates a malignant switch in breast tumor cells, leading to increased release of pro-angiogenic factors MMP-9, VEGF and TGF-β from tumor cells and intensified invasion and progression of breast cancer. Proof of concept for the crucial role played by transcription factor Fra-1 in regulating these processes was established by specific knockdown of Fra-1 with siRNA which resulted in marked suppression of tumor cell invasion, angiogenesis and metastasis in a mouse breast cancer model. Such a strategy could eventually lead to future efficacious treatments of metastatic breast cancer.

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor's stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ( - ) , AroA ( - )) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-gamma release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8+ T-cell response against Legumain+ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.

Angiogenesis is a rate-limiting step in the development of tumors. Here, we demonstrate that oral minigene DNA vaccines against murine vascular endothelial growth factor receptor-2 (FLK-1), a self-antigen overexpressed on proliferating endothelial cells in the tumor vasculature, induced protection against tumors of different origin in syngeneic BALB/c mice. This protection is mediated by CD8 T cells, which specifically kill FLK-1 + endothelial cells, resulting in marked suppression of tumor angiogenesis. More importantly, the minigene vaccine proved to be of similar efficacy as a vaccine encoding the whole FLK-1 gene. These data suggest a FLK-1 minigene vaccine provides a more flexible alternative to the whole gene vaccine, and will facilitate their future design and clinical applications in cancer therapy and prevention.